Sieving matrix

Capillary SDS-Sieving Electrophoresis In the presence of a sieving matrix, mobility decreases monotonically with molecular weight for SDS-complexed proteins. This relationship is the basis of SDS-PAGE separation of proteins. 

The precision of the assay for nonreduced samples was demonstrated by the evaluation of six independent sample preparations on a single day (repeatability) and the analysis of independent sample preparations on three separate days by two different analysts (intermediate precision). The RSD values for the migration time were 0.9%. The RSD values for peak area percent of the main peak and the minor peaks in the profile were 0.6 and 12.6%, respectively. The higher variability observed with the minor peaks was determined to be primarily related to the sample heating during preparation for the analysis. These results demonstrate that the use of uncoated fused-silica capillaries in combination with a sieving matrix can provide adequate precision and analyte recovery. 

Nowadays, the sieving matrices most employed in CSE are polymer solutions that under suitable conditions may form a transient mesh or sieving matrix that provide the size-based separation of charged biopolymers. The polymer solutions can be formulated with linear acrylamide and N-substituted acrylamide polymers, cellulose derivatives, polyethylene oxide, and its copolymers or with a variety of polymers, such as polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), and hydroxypropyl cellulose(HPC), which do not necessitate the preventive coating of the capillary wall due to their ability to dynamically coat the inner surface of the capillary, resulting in suppressed EOE and sample interactions with the capillary wall. 

Free-solution (gel-free) CE separation has been used in the detection of a target gene sequence amplified using the CPT reaction (see Chapter 9, section 9.2.1.7 for details). The intact chimeric oligonucleotide probe (fluorescein-labeled at the 5 end and biotin-labeled at the 3 end) was separated from a cleaved probe (only fluorescein-labeled at the 5 end). Owing to the presence of biotin in the intact probe, it migrated later than the cleaved probe, even in the absence of a gel sieving matrix [606]. 

Various sieving matrices for DNA fragment separation in a glass chip were evaluated. It was found the performance of HEC was comparable to PDMA, but was superior to polyacrylamide and PEO [611]. Hydroxylpropylcellulose (HPC), instead of HEC or HPMC, was used as a sieving matrix to achieve better DNA separations. In addition, the lower viscosity of HPC allows for more concentrated solution to be used without filling problems. CGE separation of DNA for analyzing mutations associated with Duchenne muscular dystrophy (DMD) was demonstrated [612],... 

DNA separation was also achieved in nanostmctured regions that were constructed by magnetic beads (700 nm). These beads were self-assembled with 5.7-pm spacings in the presence of a magnetic field. Then separation of DNA molecules (15, 33.5, and 48.5 kbp) was conducted in such an environment [993,994]. In another report, separations of X and T4 DNA was conducted in a sieving matrix formed by smaller-sized magnetic beads (570 nm) which were self-assembled in a PDMS chip [995]. 

Sanders, J.C., Breadmore, M.C., Kwok, Y.C., Horsman, K.M., Landers, J.P., Hydroxypropyl cellulose as an adsorptive coating sieving matrix for DNA separations Artificial neural network optimization for microchip analysis. Anal. Chem. 2003, 75, 986-994. 

Werner, W. E., Demorest, D. M., Stevens, J., and Wiktorowicz, J. E. (1993). Size-dependent separation of proteins denatured in SDS by capillary electrophoresis using a replaceable sieving matrix. Anal. Biochem. 212, 253-258. 

With PVA-coated capillaries [6], it was possible to separate at pH 2.5 the polyelectrolyte 2-polyvinylpyridinium hydrochloride (2-PVP) in the molecular mass range between 1.5 and 1730 kDa with dextran as sieving matrix [26]. A separation of standards is depicted in Fig. 13. Comparing the efficiencies of the... 

Phthalic acid anhydride reacts readily with alcoholic end groups of PEG. Besides an appropriate chromophore, charges are introduced simultaneously. Single- and double-labeled molecules are formed when two possible alcohohc end groups are available. The separation of derivatives of low molecular weight, such as PEG 1000, is shown in Fig. 14. Here, no sieving matrix was added. The homo-logues of the double labeled derivatives were completely separated [32]. 

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